PROJECT SUMMARY/ABSTRACT Alcohol Use Disorder (AUD) is a chronic relapsing illness associated with high rates of relapse and in which there is great need to develop and evaluate novel treatments to decrease relapse rates and the associated burden of AUD. This application proposes a novel, mechanistic combined laboratory and clinical outcome study to examine whether the neuroactive steroid precursor Pregnenolone (PREG) via its conversion to the potent GABAergic neuroactive steroid Allopregnanolone (ALLO) decreases provoked alcohol craving and anxiety, normalizes stress dysregulation, and improves cognitive flexibility and alcohol use outcomes in treatment seeking individuals with AUD. Previous research by our group and others has shown that chronic alcohol abuse dysregulates brain stress pathways including the hypothalamic pituitary adrenal (HPA) axis responses and is associated high provoked alcohol craving, anxiety, and cognitive flexibility, which in turn, are predictive of subsequent alcohol relapse and clinical outcomes. Promising new preliminary findings from our laboratory indicate that potentiating ALLO via administration of its precursors, including PREG, may reverse such stress-related disruptions and decrease alcohol craving and relapse risk. However, the mechanism by which PREG, and the specific doses at which it may potentially decrease alcohol craving and relapse risk is not known. On the basis of these findings, we propose a proof-of-concept 4-year, randomized, double-blind, dose dependent laboratory and clinical study to evaluate the preliminary efficacy of PREG treatment (200/400 mg/day for 8 weeks) versus placebo (PBO) in 90 AUD men and women. The following specific aims will be addressed: Aim 1: To evaluate the safety/tolerability of 200mg and 400mg/daily of PREG in AUD individuals. Aim 2a: To evaluate the effects of PREG doses (PBO, 200 and 400 mg/day) on ALLO levels and on experimentally provoked craving, HPA dysregulation, anxiety, mood and cognitive flexibility in AUD patients. Aim 2b: To assess whether PREG-stimulated ALLO levels mediate its effects on provoked craving, HPA dysregulation, anxiety, mood and cognitive flexibility in the laboratory component. Aim 3a: To assess the preliminary efficacy of 8-week PREG doses versus PBO treatment on primary alcohol use outcomes and secondary outcomes of alcohol craving, anxiety and negative mood. Aim 3b: To assess whether PREG- stimulated ALLO levels mediate its effects on primary alcohol use outcomes and on secondary clinical outcomes during the 8-week treatment phase. Exploratory Aim: To explore whether pre-treatment patient characteristics (gender, family history of alcoholism (FH), trauma history and co-morbid drug use) influence PREG-potentiated ALLO levels and primary and secondary alcohol use outcomes. Successful completion of the proposed aims has the potential to support further development of novel neuroactive steroid targets such as PREG and ALLO in the treatment of AUD, particularly to target chronic alcohol-related stress dysregulation, alcohol craving and high alcohol relapse risk.